CN107557453A - Primer, probe, kit and its application based on RPA technology for detection pig derived components - Google Patents
Primer, probe, kit and its application based on RPA technology for detection pig derived components Download PDFInfo
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- CN107557453A CN107557453A CN201710818666.1A CN201710818666A CN107557453A CN 107557453 A CN107557453 A CN 107557453A CN 201710818666 A CN201710818666 A CN 201710818666A CN 107557453 A CN107557453 A CN 107557453A
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Abstract
The invention discloses a kind of primer and probe combination based on RPA technology for detection pig derived components, its forward primer is as shown in SEQ ID No.1, and reverse primer is as shown in SEQ ID No.2, and probe is as shown in SEQ ID No.3.The invention also discloses the kit based on RPA technology for detection pig derived components and its application.RPA detection pig derived components are carried out using the primer and probe of the present invention, there is the characteristics of high sensitivity, high specificity and detection time are short, convenient and swift, application prospect is extensive.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of primer based on RPA technology for detection pig derived components, spy
Pin, kit and its application.
Background technology
The adulteration of meat and meat products turns into the object paid close attention in the whole world, the problem with economic quickly increase
Generation not only encroach on consumer's interests, also influence consumer to the degree of belief of businessman.
The technology species of detection animal derived materials is more and complicated at present, wherein the Protocols in Molecular Biology hand based on nucleic acid
Section mainly has quantitative real-time PCR (qPCR) and ring mediated isothermal amplification method (LAMP).
Tracking is marked to PCR primer, is existed in real time by the specific probe of fluorescence labeling for real-time fluorescence quantitative PCR
Line monitors course of reaction, product can be analyzed with reference to corresponding software, calculates the initial concentration of testing sample template.
Detection method is with the specific primer of a pair of species conservative regions, is equipped with FQ-Buffer, hot resistant DNA polymerase, four kinds of nucleosides
The compositions such as acid, amplification in vitro is carried out by qPCR instrument.However, conventional PCR processes must pass through different temperatures link, it is denatured, moves back
Fire and extension；And PCR reactions need or so 2 hours to complete, time-consuming, is unsuitable for being used for quickly detecting sample.
LAMP methods are characterized in designing two pairs of different primers for six sections on target dna chain, then recycled
Strand replacement reaction carries out amplified reaction at a certain temperature.The reaction is needed gene template, primer, strand displacement type DNA synthesis
Enzyme, matrix etc. are collectively disposed under certain temperature (60 DEG C~65 DEG C), are completed through a step.However, draw needed for the detection of LAMP methods
Thing is more and design process is complicated；Reaction temperature is higher, and the reaction time needs 60 minutes or so to complete；And easily form false positive
As a result etc..
RPA technologies are the nucleic acid amplification technologies developed in recent years, because its reaction has no special requirements and reacted to temperature
It is simple and quick, i.e., under the conditions of 37 DEG C~42 DEG C, in 15~20 minutes can quick detection target to be measured animal derived nucleic acid into
Point.Have document at present to disclose using the animal derived nucleic acid compositions of RPA technology for detection, such as patent document
CN201710379517.X discloses a kind of RPA primers, kit and detection method for detecting pig derived component；Patent document
CN201710378244.7 discloses a kind of RPA primers, kit and detection method for detecting duck derived component, but above-mentioned public affairs
For the document opened using RPA-basic methods, its defect is after reaction amplification terminates, it is also necessary to passes through agarose gel electrophoresis
Step can just obtain testing result, therefore time-consuming for whole flow process.RPA-exo technologies are compared with RPA-basic, in amplified reaction
Specific fluorescence probe is with the addition of in system, the addition of probe had both enhanced the specificity of reaction, and also achieved to reacting
The real-time monitoring of journey.
In summary, the key point based on RPA-exo technologies is the design of primer and probe.In RPA-exo amplified reactions
In, the real-time monitoring to course of reaction can be realized by the real-time collection to fluorescence signal, reaction terminates can be by generation
Amplification curve diagram judged result, and the step of no agarose gel electrophoresis, whole flow process is simple, takes short.Therefore this hair
It is bright to propose that a kind of primer, probe based on pork content in RPA-exo method quick detection meat and meat products have important meaning
Justice.
The content of the invention
In view of the defects of above-mentioned prior art is present, the purpose of the present invention is to propose to based on RPA technology for detection pigs source property into
Primer, the probe divided, realizes quick, the sensitive and specific detection to pig derived component.
The purpose of the present invention will be achieved by the following technical programs：
It is a kind of based on RPA technology for detection pig derived components primer and probe combination, including forward primer, reverse primer and
Probe, wherein, forward primer is as shown in SEQ ID No.1, and reverse primer is as shown in SEQ ID No.2, probe such as SEQ ID
Shown in No.3.
Another object of the present invention is to propose the kit based on RPA technology for detection pig derived components, easy to detect fast
Victory, application prospect are extensive.
A kind of kit based on RPA technology for detection pig derived components, including combined containing above-mentioned primer and probe
RPA- pig derived component nucleic acid detection reagents.
Preferably, above-mentioned RPA- pigs derived component nucleic acid detection reagent also includes enzyme mixation, ddH2O, rehydration buffers
Liquid.
Preferably, above-mentioned enzyme mixation includes recombinase, single strand binding protein, archaeal dna polymerase.
Mentioned reagent box also includes 280mM magnesium acetate solutions and the template DNA as positive control.
Third object of the present invention is to propose the application based on RPA technology for detection pig derived components, high sensitivity, special
Property is strong, and detection time is short, simple to operate, convenient and swift, and application prospect is extensive.
A kind of application using above-mentioned kit in based on RPA technology for detection pig derived components.
A kind of method based on RPA technology for detection pig derived components, comprises the following steps：The DNA for extracting testing sample makees
For template, the RPA amplification systems of the primer and probe according to containing claim 1, RPA amplified reactions and glimmering in real time are carried out
Light detects, and such as obtains obvious amplification curve, then proves to detect to contain pork content in sample.
Preferably, above-mentioned RPA amplification systems are：The μ L of total system 10, include the μ L of rehydration buffer solution 5.9,10 μM of forward direction
Primer 0.42 μ L, 10 μM of reverse primer 0.42 μ L, 10 μM of the μ L of 0.12 μ L, 50~200ng/ μ L template DNAs of probe 0.5,
ddH2O 0.14 μ L, 280mM the μ L of magnesium acetate solution 0.5, the μ L of enzyme mixation 2.
Preferably, above-mentioned RPA amplified reactions are 37 DEG C~42 DEG C of temperature, are reacted 15~20 minutes.
Preferably, above-mentioned RPA amplified reactions are carried out in real-time fluorescence detector.
Compared with prior art, a kind of primer based on RPA technology for detection pig derived components provided by the invention, probe,
Kit and its application, what is reached has the technical effect that：1) relative to qPCR technologies, the invention has the advantages that：A. at 39 DEG C
RPA amplified reactions can be carried out, or even body temperature can provide the temperature environment required for RPA amplified reactions, its most thermophilic
Degree is at 37 DEG C~42 DEG C, and whole amplified reaction process is isothermal, and standard PCR amplification reaction has to pass through different temperatures
Set；B. detection time is short：Whole detection process can be completed at 15 minutes or so, and without denaturation, not only substantially reduce
The reaction time is detected, without the special temperature control device similar with PCR instrument, it is achieved thereby that portable Rapid nucleic acid detects；
The characteristics of c.RPA technologies also maintain high specific and high sensitivity while the reaction time is shortened；
2) relative to LAMP methods, the invention has the advantages that：A. both are all isothermal amplification, and RPA is reacted 37
DEG C~42 DEG C can carry out RPA amplified reactions, or even the temperature environment required for body temperature can provide reaction, and LAMP
60 DEG C~65 DEG C of the temperature requirement of method, temperature requirement is higher；B.RPA reactions need to only design pair of primers and a fluorescence is visited
Pin, while reaction sensitivity is ensured, the specificity of reaction is also ensure that, and LAMP reactions need to design two pairs of primers, draw
Thing design is got up more complicated；
3) present invention detects the pig derived component in meat and meat products, has high sensitivity, high specificity, detection time short
And the features such as simple to operate, convenient and swift, application prospect is extensive.
Below just in conjunction with the embodiments and accompanying drawing, the embodiment of the present invention is described in further detail, so that technology
Scheme is more readily understood, grasped.
Brief description of the drawings
Fig. 1 is RPA amplifications principle schematic of the present invention in embodiment 1；
Fig. 2 is fluorescence probe operation principle schematic diagram of the present invention in embodiment 1；
Fig. 3 is the specificity figure that the primer designed and probe of the invention are verified in embodiment 4.
Embodiment
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.In following embodiments
The experimental method, it is conventional method unless otherwise specified；The reagent and material, unless otherwise specified, can be from business
Approach obtains, example below and the scope of the claims for being not used to the limitation present invention, all equivalence enforcements without departing from carried out by the present invention
Or change, it is intended to be limited solely by the scope of this patent.
The design and screening of embodiment 1 RPA primers, probe
RPA amplifications principle is as shown in Figure 1：, it is necessary to which the participation of three kinds of enzymes, is recombinase, single-stranded knot respectively in RPA reactions
Hop protein and archaeal dna polymerase.When RPA is expanded, recombinase is combined with upstream and downstream primer first, finds homologous double-stranded DNA,
Once positioning, chain occurring and exchanges, single strand binding protein is bound with parent's chain, prevents the template strand of itself and disengaging that phase interaction occurs
With；Then, archaeal dna polymerase starts templated synthesis from 3 ' ends of upstream and downstream primer, forms two double-stranded DNAs, so moves in circles
And then realize amplification.
Fluorescence probe operation principle is as shown in Figure 2：A specificity is added when RPA is expanded while pair of primers is added
Fluorescence probe, the probe is an oligonucleotides, respectively one fluorescent reporter group of mark and a fluorescent quenching group.
When probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions；When RPA is expanded, exo nucleases
Endonuclease activity cuts tetrahydrofuran (THF) site on probe, separates fluorescent reporter group and fluorescent quenching group, from
And fluorescence detecting system can receive fluorescence signal, the accumulation and RPA products that realize fluorescence signal form Complete Synchronization.
To realize the specificity of RPA detections, it is necessary to search out the specific DNA sequence dna that pig is different from other species, if
Specificity amplification primer pair is counted out, while designs the fluorescence probe that can be specifically bound with amplified production.
The present invention consults by lot of documents early stage, first according in qPCR or LAMP technology shown in consulting literatures
The primer of pig derived component design is detected, then selectes specific detection gene, and designs the primer and probe of the present invention, most
The primer to design, probe carry out RPA-exo experiments afterwards, identify specificity, the sensitivity of its reaction.
The primer and probe for the detection pig derived component that the present invention finally gives are as shown in table 1：Forward primer sequence such as SEQ
Shown in ID No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
Primer and probe sequence of the table 1 based on RPA technology for detection pig derived components
Fluorescent reporter group, fluorescent quenching group, the position in tetrahydrofuran site wherein in probe sequence is specific as follows：
GGG GAA CAG CAG GAG(FAM-dT)(THF)(BHQ1-dT)GGG ACG GAA ACA AAT CAG CAG ATG AAA
GGA(phosphate)；
Wherein,：FAM：Luminophore；THF：Tetrahydrofuran site；BHQ1：Quenching group；phosphate：Phosphate group.
Kit of the embodiment 2 based on RPA technology for detection pig derived components
The kit of the present invention includes RPA- pig derived components nucleic acid detection reagent, 280mM magnesium acetate solutions, pig standard matter
Grain (positive control)；Wherein RPA- pigs derived component nucleic acid detection reagent includes forward primer, reverse primer, probe, ddH2O、
Enzyme mixation (recombinase, single strand binding protein, archaeal dna polymerase), rehydration buffer solution.
The nucleic acid amplification of embodiment 3
RPA amplification systems form：The μ L of total system 10, include RPA- pig derived components nucleic acid detection reagent 9 μ L, 280mM
The μ L of 0.5 μ L, 50~200ng/ μ L template DNAs of magnesium acetate solution 0.5；
Wherein, 9 μ L RPA- pig derived component nucleic acid detection reagents include：The μ L of rehydration buffer solution 5.9,10 μM of forward direction
Primer 0.42 μ L, 10 μM of reverse primer 0.42 μ L, 10 μM of the μ L of probe 0.12, enzyme mixation 2 μ L, ddH2O 0.14μL；
Above-mentioned enzyme mixation includes recombinase, single strand binding protein and archaeal dna polymerase etc..
RPA amplification programs are：39 DEG C of temperature, react 15 minutes.
The RPA amplified reactions are carried out in fluorescence detector.
The primer of the checking design of embodiment 4 and the specificity of probe
1. reagent and instrument
RPA- pig derived components nucleic acid detection reagent, 280mM magnesium acetate solution, the standard plasmid of pig are (as positive right
According to).Wherein RPA- pigs derived component nucleic acid detection reagent contains enzyme mixation (recombinase, single strand binding protein, DNA polymerizations
Enzyme), forward primer, reverse primer, probe, rehydration buffer solution.
Wherein, primer pair and the sequence of probe are as follows：
Fluorescent reporter group, fluorescent quenching group, the position in tetrahydrofuran site wherein in probe sequence is specific as follows：
GGG GAA CAG CAG GAG(FAM-dT)(THF)(BHQ1-dT)GGG ACG GAA ACA AAT CAG CAG ATG AAA
GGA(phosphate)；
Wherein, FAM：Luminophore；THF：Tetrahydrofuran site；BHQ1：Quenching group；phosphate：Phosphate group；
RNase-free Water (Beijing Quanshijin Biotechnology Co., Ltd)；BDA6 fluorescence detectors；Pipettor；Whirlpool
Revolve shaker；Enzyme mixation is purchased from TwistDX companies, entitled core reaction mixture (core reaction mix)；
2nd, sample
The sample be Suzhou Borda bio tech ltd laboratory preserve using post formulation extract respectively chicken,
Duck, goose, pig, ox, sheep, horse, fox, the DNA of mouse.
3rd, experimental method
3.1 sample preparation
The DNA10 μ L of goose, chicken, duck, ox, sheep, horse, fox, mouse are taken respectively in a sterilized 1.5mL centrifuge tube
In, it is vortexed and mixes, obtains DNA mixed liquors, be named as pig M；
The DNA10 μ L of pig are taken in a sterilized 1.5mL centrifuge tube, is vortexed and mixes, target sample nucleic acid is obtained and carries
Thing is taken, is named as pig DNA extract.
3.2 operating procedure
Four groups are set to, is respectively：A. negative control group：Take 9 μ L RPA- pig derived component nucleic acid detection reagents, respectively according to
0.5 μ LddH of secondary addition2O, 0.5 μ L magnesium acetate solutions (280mM), through centrifugation 5 seconds-vibration 10 seconds-centrifugation 5 seconds, negative control is obtained
Group；
B. positive controls：9 μ L RPA- pig derived component nucleic acid detection reagents are taken, are sequentially added into 0.5 μ L pig standards
Plasmid, 0.5 μ L magnesium acetate solutions (280mM), through centrifugation 5 seconds-vibration 10 seconds-centrifugation 5 seconds, obtain positive controls；
C. pig DNA extract group：9 μ L RPA- pig derived component nucleic acid detection reagents are taken, are sequentially added into 0.5 μ L pigs
DNA extracts (66ng/ μ L), 0.5 μ L magnesium acetate solutions (280mM), through centrifugation 5 seconds-vibration 10 seconds-centrifugation 5 seconds, pig DNA carries
Take thing group；
D. pig M groups：9 μ L RPA- pig derived component nucleic acid detection reagents are taken, are sequentially added into 0.5 μ L pigs M, 0.5 μ L vinegar
Sour magnesium solution (280mM), through centrifugation 5 seconds-vibration 10 seconds-centrifugation 5 seconds, obtain pig M groups；
Above-mentioned four groups of reaction tubes are put into fluorescence detector and carry out amplified reaction, 15 minutes reaction time, reaction temperature
39 DEG C, while the amplification curve of Real Time Observation reaction carries out result judgement, such as obtains obvious amplification curve, then proves to be detected
Contain pork content in sample.
4th, experimental result
The pig specific forward primer and reverse primer designed using the present invention, to negative control, positive control, pig DNA
Extract, pig M carry out RPA amplifications and real-time fluorescence detection, can rapidly and accurately identify pork content.Wherein, in the positive
There is obvious amplification curve in control and pork extract DNA, and there is no amplification curve in negative control and pig M, detection knot
Fruit is as shown in Figure 3.
Some preferred embodiments of the present invention have shown and described in described above, but as previously described, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations,
Modification and environment, and above-mentioned teaching or the technology or knowledge of association area can be passed through in the scope of the invention is set forth herein
It is modified., then all should be in this hair and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of the present invention
In the protection domain of bright appended claims.
Sequence table
<110>Suzhou Borda bio tech ltd
<120>Primer, probe, kit and its application based on RPA technology for detection pig derived components
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tatcagcccc catctaccaa caagaaaacg aa 32
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctcgctgtct gcttctctct catcctcctt gc 32
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ggggaacagc aggaggggac ggaaacaaat cagcagatga aagga 45
Claims (10)
1. a kind of primer and probe combination based on RPA technology for detection pig derived components, including forward primer, reverse primer and spy
Pin, it is characterised in that the forward primer is as shown in SEQ ID No.1, and the reverse primer is as shown in SEQ ID No.2, institute
Probe is stated as shown in SEQ ID No.3.
2. a kind of kit based on RPA technology for detection pig derived components, it is characterised in that including containing described in claim 1
Primer and probe combination RPA- pig derived component nucleic acid detection reagents.
3. kit according to claim 2, it is characterised in that the RPA- pigs derived component nucleic acid detection reagent also wraps
Include enzyme mixation, ddH2O, rehydration buffer solution.
4. kit according to claim 3, it is characterised in that the enzyme mixation includes recombinase, single-stranded combination egg
In vain, archaeal dna polymerase.
5. kit according to claim 2, it is characterised in that the kit also include 280mM magnesium acetate solutions and
Template DNA as positive control.
A kind of 6. kit using described in claim any one of 2-5 answering in based on RPA technology for detection pig derived components
With.
A kind of 7. method based on RPA technology for detection pig derived components, it is characterised in that comprise the following steps：Test sample is treated in extraction
The DNA of product the RPA amplification systems of the primer and probe according to containing claim 1, it is anti-to carry out RPA amplifications as template
Answer and real-time fluorescence detects, such as obtain obvious amplification curve, then prove to detect to contain pork content in sample.
8. according to the method for claim 7, it is characterised in that the RPA amplification systems are：The μ L of total system 10, comprising again
Rehydration buffer 5.9 μ L, 10 μM of forward primer 0.42 μ L, 10 μM of reverse primer 0.42 μ L, 10 μM of probe 0.12 μ L, 50
~200ng/ μ L template DNAs 0.5 μ L, ddH2O 0.14 μ L, 280mM the μ L of magnesium acetate solution 0.5, the μ L of enzyme mixation 2.
9. according to the method for claim 7, it is characterised in that the RPA amplified reactions are 37 DEG C~42 DEG C of temperature, reaction
15~20 minutes.
10. according to the method for claim 7, it is characterised in that the RPA amplified reactions enter in real-time fluorescence detector
OK.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710818666.1A CN107557453A (en) | 2017-09-12 | 2017-09-12 | Primer, probe, kit and its application based on RPA technology for detection pig derived components |
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| CN201710818666.1A CN107557453A (en) | 2017-09-12 | 2017-09-12 | Primer, probe, kit and its application based on RPA technology for detection pig derived components |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108118086A (en) * | 2018-01-30 | 2018-06-05 | 西北民族大学 | For detecting RPA primers, probe and the method for pork content in meat products |
| CN113969320A (en) * | 2021-09-26 | 2022-01-25 | 华南农业大学 | RPA primer for identifying pig-derived components, detection system and method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108118086A (en) * | 2018-01-30 | 2018-06-05 | 西北民族大学 | For detecting RPA primers, probe and the method for pork content in meat products |
| CN113969320A (en) * | 2021-09-26 | 2022-01-25 | 华南农业大学 | RPA primer for identifying pig-derived components, detection system and method |
| CN113969320B (en) * | 2021-09-26 | 2023-07-25 | 华南农业大学 | RPA primer for identifying swine-derived components, detection system and detection method |
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